This invention claims priority of the German patent application 100 56 382.1 which is incorporated by reference herein.
The invention concerns a light source for illumination in scanning microscopy.
The invention further concerns a scanning microscope. The scanning microscope can also be configured as a confocal microscope.
In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of the illuminating light beam is moved in a specimen plane with the aid of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually at right angles to one another, so that one mirror deflects in the X and the other in the Y direction. The tilting of the mirrors is brought about, for example, using galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors for ascertaining the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the excitation stop), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen arrives via the beam deflection device back at the beam splitter, passes through it, and is then focused onto the detection stop behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, so that a point datum is obtained which, by sequential scanning of the specimen, results in a three-dimensional image. Usually a three-dimensional image is obtained by image acquisition in layers.
The power level of the light coming from the specimen is measured at fixed time intervals during the scanning operation, and thus scanned one grid point at a time. The measured value must be unequivocally associated with the pertinent scan position so that an image can be generated from the measured data. Preferably, for this purpose the status data of the adjusting elements of the beam deflection device are also continuously measured, or (although this is less accurate) the setpoint control data of the beam deflection device are used.
In a transmitted-light arrangement it is also possible, for example, to detect the fluorescent light, or the transmission of the exciting light, on the condenser side. The detected light beam does not then pass via the scanning mirror to the detector (non-descan configuration). For detection of the fluorescent light in the transmitted-light arrangement, a condenser-side detection stop would be necessary in order to achieve three-dimensional resolution as in the case of the descan configuration described. In the case of two-photon excitation, however, a condenser-side detection stop can be omitted, since the excitation probability depends on the square of the photon density (i.e., excitation probability is proportional to intensity2), which of course is much greater at the focus than in neighboring regions. The fluorescent light to be detected therefore derives, with high probability, almost exclusively from the focus region; this makes superfluous any further differentiation between fluorescent photons from the focus region and fluorescent photons from the neighboring regions using a stop arrangement.
The resolution capability of a confocal scanning microscope is determined, among other factors, by the intensity distribution and spatial extension of the focus of the illuminating light beam. An arrangement for increasing the resolution capability for fluorescence applications is known from PCT/DE/95/00124. In this, the lateral edge regions of the illumination focus volume are illuminated with light of a different wavelength that is emitted by a second laser, so that the specimen regions excited there by the light of the first laser are brought back to the ground state in stimulated fashion. Only the light spontaneously emitted from the regions not illuminated by the second laser is then detected, the overall result being an improvement in resolution. This method has become known as STED (stimulated emission depletion).
Two lasers are usually used in STED microscopy, i.e. one to excite a specimen region and another to generate the stimulated emission. In particular for generating the stimulated emission, high light outputs and at the same time a maximally flexible wavelength selection are needed. Optical parametric oscillators (OPOs) are often used for this purpose. OPOs are very expensive, and moreover require high-powered pumping lasers. These are usually mode-coupled pulsed lasers, which are also very expensive. Costs for the exciting light source must also be added. All the lasers must furthermore be exactly aligned so as to arrive exactly at the individual specimen regions. In the case of pulsed excitation, it is important for the light pulses generating the stimulated emission to arrive within a specific time framexe2x80x94which depends on the lifetime of the excited states of the specimen materialxe2x80x94after the exciting light pulses. Synchronizing the pulsed lasers with one another is complex, and the result is often unsatisfactory and unstable.
It is the object of the invention to create a light source for illumination in scanning microscopy which is easy to handle, reliable, flexible and allows for STED microscopy in a less expensive way.
This object is achieved by a light source for illumination in scanning microscopy comprising:
an electromagnetic energy source that emits light of one wavelength,
a means for spatially dividing the light into at least two partial light beams, which is placed after the electromagnetic energy source; and
an intermediate element for wavelength modification in at least one partial light beam.
A further object of the invention is to create an a scanning microscope which provides a flexible, reliable and easy to handle illumination and which allows for STED microscopy in a less expensive way.
The further object is achieved by a scanning microscope comprising:
an electromagnetic energy source that emits light of one wavelength,
a means for spatially dividing the light into at least two partial light beams, which is placed after the electromagnetic energy source,
an intermediate element for wavelength modification in at least one partial light beam,
a beam deflection device for guiding the two partial light beams over a specimen and
a microscope optical system for focusing the partial light beams.
The use of the light source according to the present invention makes the illumination system for microscopy, and in particular STED microscopy, much simpler and much less expensive, since only one electromagnetic energy source is required.
In a particular embodiment, one partial light beam serves for optical excitation of a first region of a specimen. A further partial light beam, whose wavelength is modified with the aid of an intermediate element, is used to generate the stimulated emission in a further region of the specimen. The first region and the further region overlap at least partially. The wavelength of the second partial light beam is modified with an intermediate element. This intermediate element is preferably an optical parametric oscillator (OPO).
The invention has the further advantage that in the case of pulsed excitation, for example for purposes of multi-photon excitation, it is possible to dispense with synchronization among the pulsed light sources if the electromagnetic energy source that causes both the excitation and the stimulated emission is a pulsed laser.